Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
PCR technologies for quantification of gene expression have improved through the development of rapid thermocylers and the introduction of fluorescence monitoring of amplified products after each cycle (real-time PCR). Quantification of gene expression occurs through the use of dyes, particularly fluorescent dyes, and the detection of increasing fluorescence during the exponential phase of PCR amplification proportional to the amount of nucleic acids in the sample at the beginning of the reaction. Quantification is based on the threshold cycle, i.e. the first cycle with detectable fluorescence, and can be performed in absolute manner with external standards (usually a synthetic gene) or in relative manner with a comparative normalizing reference gene serving as an internal calibrator (i.e. housekeeping gene). Control genes or housekeeping genes are used to normalise mRNA levels between different samples.
It is critical that the selected reference gene does not fluctuate since even marginal variations in gene expression will alter the relative quantification profile of the target gene. Pipetting and dilution errors also alter the level of amplification and thus alter the quantification profile.
Genes such as glyceraldehyde -3-phosphate dehydrogenase (GAPDH), porphobilinogen desaminase (PBGD), beta 2-microglobin or beta-actin are often used as internal calibrators in real-time PCR. However, the aforementioned genes have been shown to move in response to experimental conditions or treatments. Genes that are abundantly expressed, such as 18S, are also not ideal reference genes as PCR conditions need to be restricted so as not to swamp the reaction.
Thus, suitable housekeeping genes should be adequately expressed in the tissue of interest, and most importantly, show minimal variability in expression between the samples and under the experimental conditions or treatments used.
Many of these control genes however can show unacceptable variability in expression. It has been shown that the expression level of such genes may vary among tissues or cells and may change under certain circumstances i.e. different treatments. Thus it is crucial to validate housekeeping genes in any new experimental system. It is often a time consuming and difficult task to find a housekeeping gene or reference gene that is suitable for use in a specific experimental system. In some situations this may not be possible.
The use of external standards (i.e. a synthetic sequence) in gene expression studies generally requires that the gene of interest be cloned to provide the synthetic reference gene. In this method, known amounts of the synthetic reference gene sequence are serially diluted then subjected to amplification to produce a standard curve. Production of the cloned sequence for this method is generally a time consuming, labour intensive task and dilution errors are amplified exponentially which can lead to inaccurate assessment of nucleic acid levels. Stability and preservation of highly diluted cloned sequences can also cause difficulties.
Thus, there remains a need for a quick and efficient universal method of quantifying nucleic acids, that is applicable to any experimental situation or treatment condition, that does not require the use of a housekeeping gene or a synthetic gene of interest to normalise data.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.